Guinea pig mesenteric lymphatic vessels exhibit rhythmic constrictions induced by action potential (AP)-like spikes and initiated by entrainment of spontaneous transient depolarizations (STDs). To characterize STDs and the signaling mechanisms responsible for their occurrence, we used intracellular microelectrodes, Ca²⁺ imaging, and pharmacological agents. In our investigation of the role of intracellular Ca²⁺ released from Ca²⁺ stores, we observed that intracellular Ca²⁺ transients accompanied some STDs, although there were many exceptions where Ca²⁺ transients occurred without accompanying STDs. STD frequency and amplitude were markedly affected by activators/inhibitors of inositol 1,4,5-trisphosphate receptors (IP₃Rs) but not by treatments known to alter Ca²⁺ release via ryanodine receptors. A role for Ca²⁺-activated Cl⁻ (ClCa) channels was indicated, as STDs were dependent on the Cl⁻ but not Na⁺ concentration of the superfusing solution and were inhibited by the ClCa channel blockers niflumic acid (NFA), anthracene 9-carboxylic acid, and 5-nitro-2-(3-phenylpropylamino)benzoic acid but not by the volume-regulated Cl⁻ blocker DIDS. Increases in STD frequency and amplitude induced by agonist stimulation were also inhibited by NFA. Nifedipine, the hyperpolarization-activated inward current blocker ZD-7288, and the nonselective cation/store-operated channel blockers SKF-96365, Gd³⁺, and Ni²⁺ had no or marginal effects on STD activity. However, nifedipine, 2-aminoethoxydiphenyl borate, NFA, SKF-96365, Gd³⁺, and Ni²⁺ altered the occurrence of spontaneous APs. Our findings support a role for Ca²⁺ release through IP₃Rs and a resultant opening of ClCa channels in STD generation and confirm the importance of these events in the initiation of lymphatic spontaneous APs and subsequent contractions. The abolition of spontaneous APs by blockers of other excitatory ion channels suggests a contribution of these conductances to lymphatic pacemaking.